Although it has long been recognized that the coagulation system plays a role in modulating inflammation, it is only recently that the impact of this contribution has been appreciated and that some of the molecular links have been established. In this respect, the protein C anticoagulant pathway is particularly relevant. In addition to its well-characterized role in modulating thrombin generation, this system, composed of a complex of soluble and membrane-associated proteins, plays an integral part in regulating the response to selected inflammatory agents (reviewed in44). Substantial clinical data have revealed that patients with severe sepsis have significantly diminished levels of protein C and protein S, and the extent of suppression of protein C may correlate with clinical outcome49. Activated protein C (APC) appears to modulate the inflammatory response by several mechanisms, including inhibiting polymorphonuclear cell (PMN) activation and elastase release, blocking PMN interactions with selecting, and preventing cytokine release by monocytes48,52-55. More recently, the endothelial cell protein receptor (EPCR), a cofactor that enhances activation of protein C by thrombin-thrombomodulin, has also been found to modulate the function of APC in inflammation. Moreover, inhibition of the interaction of APC/PC with EPCR in vivo resulted in an increased inflammatory response following E. coli infusions in baboons56. Further links between EPCR and inflammation, although not yet fully delineated, are being explored as Esmon and coworkers have reported that a soluble form of EPCR is released during sepsis57, interferes with activation of protein C, and binds to a receptor on activated neutrophils that is the autoantigen in Wegener's granulomatosis58,59. Another particularly relevant player in the anticoagulant system is thrombomodulin (TM), a critical cofactor in the activation of protein C, and a widely expressed glycoprotein receptor for thrombin. With the cloning and sequencing of the gene for thrombomodulin1, the putative structural organization of the protein and the regions responsible for its anticoagulant and anti-fibrinolytic function have been elucidated. Mature single-chain TM in the human is 557 amino acids long and is structurally divided into five domains. The N-terminal region (residues 1-226) 2 has a module (residues 1-154) with homology to the lectin domains of the hepatic asialoglycoprotein receptor and IgE, as well as to members of the selectin family. Although controversial, in vitro analyses suggest that this domain is required for constitutive internalization of the receptor in some cells5,6. From residues 155 to 226, there is a hydrophobic region which may be associated with the plasma membrane and which contains two potential sites for O-linked glycosylation. The next domain is comprised of six epidermal growth factor (EGF)-like repeats, the last 3 or 4 of which are necessary for activation of TAFI or protein C, respectively, by thrombin. The function of the other EGF-like repeats is unknown. The third domain between the EGF-like repeats and the membrane-spanning region is serine/threonine rich and contains four potential sites for O-linked glycosylation, to one of which is attached a chondroitin sulfate, important for full anticoagulant activity of TM. Fourthly, there is a highly conserved transmembrane domain, and fifthly a short cytoplasmic tail that contains potential sites of phosphorylation, and a single cysteine that may mediate multimerization of the molecule. It has been shown that TM is important in regulating the inflammatory process via the anticoagulant pathway. The downregulation of vascular endothelial cell TM by inflammatory cytokines—an effect mirrored by the expression of cellular EPCR—directly impairs the generation of APC. The protein C co-factor function of TM is also impaired in the face of inflammation, as activated PMNs release lysosomal proteases and oxidants that result in proteolysis of the receptor and oxidation of a critical methionine within the EGF-like repeats of TM that inactivates the function of glycoprotein for protein C activation. Several additional lines of evidence support a role for TM as an anti-inflammatory agent. Recombinant soluble forms of TM, most of which were composed of the entire extramembranous regions, were used to prevent endotoxin-induced pulmonary accumulation of leukocytes and ARDS, organ failure, or lethality in small animal models54,63,64. Adenovirus-mediated gene transfer of TM in a rabbit restenosis model was not only effective in reducing restenosis, but also resulted in decreased inflammation and extravasation of leukocytes22. In a spinal cord compression-induced injury model in rats, recombinant soluble TM provided neuroprotection, with reduction in leukocyte accumulation and cytokine mRNA expression65. In each of these studies, the improved outcomes following TM administration were attributed to enhanced activation of protein C, while the possibility that other domains of TM might contribute to the apparent anti-inflammatory effect was never considered. In the present invention we have determined the in vivo function of the N-terminal lectin-like domain of TM by generating mice lacking this domain and we have shown that addition of the recombinant N-terminal lectin-like domain provides the vascular endothelium with natural anti-inflammatory properties by interfering with leukocyte adhesion. (1) TM is a known molecule, (2) the EGF-regions of TM are known to have anti-coagulant (and indirectly anti-inflammatory) activity. However, the current invention surprisingly demonstrates that the lectin-like region of TM has an anti-inflammatory function. Indeed, since it has been shown in the art that several members of the C-type lectin family (to which the lectin-like domain of TM belongs) function to enhance leukocyte adhesion one would expect that the lectin-like domain of TM has rather a pro-inflammatory function.
Table 1: Response of mice exposed to hypoxia. Lung tissue levels of fibrin and plasma levels of FPA with associated SD. No significant differences between TMLeD/LeD and TMwt/wt mice were demonstrated (p>0.1).
Table 2: Response of mice exposed to LPS. Lung tissue levels of fibrin with associated SD. No significant differences between TMLeD/LeD and TMwt/wt mice were demonstrated (p>0.1).
Table 3: Response of mice exposed to sublethal dose of LPS. Serum cytokine levels were measured, as were peripheral white blood cell (WBC) counts. TNFα and IL-1β levels are significantly higher in TMLeD/LeD and TMLeDneo/LeDneo mice. For each group, n=18.
Table 4: Myeloperoxidase (MPO) activity in BALF after LPS inhalation. MPO activity is significantly higher in lungs of TMLeD/LeD mice after LPS exposure. Results are representative of an experiment performed twice.
Table 5: Plasma levels of human protein C (hPC) and human activated protein C (hAPC) following infusion of hPC as described in methods. The results reflect one of two representative experiments, each of which had 5 mice in each group.
Table 6: Bone marrow derived PMNs from either genotype mice were assessed for adhesion to fEND.5 cells in a flow chamber model. Results reflect results of 5 independent experiments. For each experiment, 15 microscopic fields were counted as detailed in methods.
Table 7: Bone marrow derived PMNs from either genotype mice were assessed for adhesion to fEND.5 cells in a flow chamber model. Results reflect results of 5 independent experiments. For each experiment, 15 microscopic fields were counted as detailed in methods.
Table 8: Static adhesion assay. PMN and lymphocyte adhesion was significantly greater to non-TNF treated TMLeD/LeD endothelial cells than to TMwt/wt endothelial cells (p<0.005). Anti-TM antisera (ab) increased PMN adhesion in TMwt/wt endothelial cells (p<0.005), but had no additional effect on PMN adhesion to TMLeD/LeD endothelial cells. This is a representative experiment performed 3 times, on 3 different clones each. For each experiment, 5 wells were used for each condition, and adherent leukocytes in 15 microscopic fields were counted.
Table 9: Effect of recombinant TMlec155 on PMN adhesion. PMNs were derived from wild-type mice. TMlec155 significantly decreased PMN adhesion to TMLeD/LeD endothelial cells.
Table 10: Effect of recombinant TMlec155 on cytokine response in vivo. Wild-type mice were treated with LPS 20 μg/gm i.p., following 5 min later with the noted treatment. Plasma levels of IL-1b were measured 3 hours later